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Channelrhodopsins (ChRs) are microbial light-gated ion channels with a retinal chromophore and are widely utilized in optogenetics to precisely control neuronal activity with light. Despite increasing understanding of their structures and photoactivation kinetics, the atomistic mechanism of light gating and ion conduction remains elusive. Here, we present an atomic structural model of a chimeric ChR in a precursor state of the channel opening determined by an accurate hybrid molecular simulation technique and a statistical theory of internal water distribution. The photoactivated structure features extensive tilt of the chromophore accompanied by redistribution of water molecules in its binding pocket, which is absent in previously known photoactivated structures of analogous photoreceptors, and widely agrees with structural and spectroscopic experimental evidence of ChRs. The atomistic model manifests a photoactivated ion-conduction pathway that is markedly different from a previously proposed one and successfully explains experimentally observed mutagenic effects on key channel properties.

The triose-phosphate/phosphate translocator (TPT) catalyses the strict 1:1 exchange of triose-phosphate, 3-phosphoglycerate and inorganic phosphate across the chloroplast envelope, and plays crucial roles in photosynthesis. Despite rigorous study for more than 40 years, the molecular mechanism of TPT is poorly understood because of the lack of structural information. Here we report crystal structures of TPT bound to two different substrates, 3-phosphoglycerate and inorganic phosphate, in occluded conformations. The structures reveal that TPT adopts a 10-transmembrane drug/metabolite transporter fold. Both substrates are bound within the same central pocket, where conserved lysine, arginine and tyrosine residues recognize the shared phosphate group. A structural comparison with the outward-open conformation of the bacterial drug/metabolite transporter suggests a rocker-switch motion of helix bundles, and molecular dynamics simulations support a model in which this rocker-switch motion is tightly coupled to the substrate binding, to ensure strict 1:1 exchange. These results reveal the unique mechanism of sugar phosphate/phosphate exchange by TPT. The first crystal structures of TPT, a membrane transporter that exports the Calvin cycle intermediates from chloroplasts and plays fundamental roles in nearly all photosynthetic eukaryotes, have now been resolved in complex with different substrates.

Ca2+release from the sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) is crucial for muscle contraction, cell growth, apoptosis, learning and memory. The trimeric intracellular cation (TRIC) channels were recently identified as cation channels balancing the SR and ER membrane potentials, and are implicated in Ca2+signaling and homeostasis. Here we present the crystal structures of prokaryotic TRIC channels in the closed state and structure-based functional analyses of prokaryotic and eukaryotic TRIC channels. Each trimer subunit consists of seven transmembrane (TM) helices with two inverted repeated regions. The electrophysiological, biochemical and biophysical analyses revealed that TRIC channels possess an ion-conducting pore within each subunit, and that the trimer formation contributes to the stability of the protein. The symmetrically related TM2 and TM5 helices are kinked at the conserved glycine clusters, and these kinks are important for the channel activity. Furthermore, the kinks of the TM2 and TM5 helices generate lateral fenestrations at each subunit interface. Unexpectedly, these lateral fenestrations are occupied with lipid molecules. This study provides the structural and functional framework for the molecular mechanism of this ion channel superfamily.

The drug/metabolite transporter (DMT) superfamily is a large group of membrane transporters ubiquitously found in eukaryotes, bacteria and archaea, and includes exporters for a remarkably wide range of substrates, such as toxic compounds and metabolites. YddG is a bacterial DMT protein that expels aromatic amino acids and exogenous toxic compounds, thereby contributing to cellular homeostasis. Here we present structural and functional analyses of YddG. Using liposome-based analyses, we show that Escherichia coli and Starkeya novella YddG export various amino acids. The crystal structure of S. novella YddG at 2.4 Å resolution reveals a new membrane transporter topology, with ten transmembrane segments in an outward-facing state. The overall structure is basket-shaped, with a large substrate-binding cavity at the centre of the molecule, and is composed of inverted structural repeats related by two-fold pseudo-symmetry. On the basis of this intramolecular symmetry, we propose a structural model for the inward-facing state and a mechanism of the conformational change for substrate transport, which we confirmed by biochemical analyses. These findings provide a structural basis for the mechanism of transport of DMT superfamily proteins.

P2X receptors are trimeric ATP-gated cation channels involved in physiological processes ranging widely from neurotransmission to pain and taste signal transduction. The modulation of the channel gating, including that by divalent cations, contributes to these diverse physiological functions of P2X receptors. Here, we report the crystal structure of an invertebrate P2X receptor from the Gulf Coast tick Amblyomma maculatumin the presence of ATP and Zn2+ion, together with electrophysiological and computational analyses. The structure revealed two distinct metal binding sites, M1 and M2, in the extracellular region. The M1 site, located at the trimer interface, is responsible for Zn2+potentiation by facilitating the structural change of the extracellular domain for pore opening. In contrast, the M2 site, coupled with the ATP binding site, might contribute to regulation by Mg2+. Overall, our work provides structural insights into the divalent cation modulations of P2X receptors.

The bacterial SecYEG translocon functions as a conserved protein-conducting channel. Conformational transitions of SecYEG allow protein translocation across the membrane without perturbation of membrane permeability. Here, we report the crystal structures of intact SecYEG at 2.7-Å resolution and of peptide-bound SecYEG at 3.6-Å resolution. The higher-resolution structure revealed that the cytoplasmic loop of SecG covers the hourglass-shaped channel, which was confirmed to also occur in the membrane by disulfide bond formation analysis and molecular dynamics simulation. The cytoplasmic loop may be involved in protein translocation. In addition, the previously unknown peptide-bound crystal structure of SecYEG implies that interactions between the cytoplasmic side of SecY and signal peptides are related to lateral gate opening at the first step of protein translocation. These SecYEG structures therefore provide a number of structural insights into the Sec machinery for further study.